PROJECT SUMMARY/ABSTRACT Acinetobacter baumannii is one of the most drug resistant bacterial pathogens. Blood or lung infections caused by extreme drug resistant (XDR) A. baumannii have 50% mortality rates, and the antimicrobial pipeline for such infections is inadequate. We screened 11,862 compounds against A. baumannii strains that were cultured in RPMI with serum as a nutrient-depleted media to mimic in vivo conditions, rather than nutrient-rich MHII. In RPMI, rifabutin (RBT) was 133-fold more active than rifampin (RIF) against A. baumannii, with MICs of 0.031 g/ml and 4 g/ml respectively. However, no difference in activity was observed when MHII was used as the culture media. Furthermore, in preliminary studies, RBT was superior to RIF in our murine models of lethal XDR A. baumannii infections. Finally, spiking MHII media into RPMI also reversed the increased activity of RBT, indicating that some component of MHII actively antagonizes the RBT. In our preliminary data, RBT is more effective than RIF in vitro and in preliminary mouse studies. The goal of this proposal is to determine the translational potential of a fundamental modification of antibacterial screening methodologies by defining the basis of enhanced efficacy of RBT vs. RIF as a treatment for A. baumannii, and defining RBT efficacy in pneumonia and bacteremia. Specific Aim 1: Define the mechanistic driver of RBT vs. RIF differential efficacy. MHII media will be fractionated by size and/or chromatography. The composition of the fractions that inhibit RBT activity will be defined by LC-MS/MS. Specific Aim 2: Defining the relative in vivo efficacy of RBT vs. RIF in murine models of A. baumannii pneumonia and bacteremia with and without other antibiotic therapy. RBT will be dose optimized to maximize improvement in survival. Treatment with RBT at multiple doses will be compared to placebo, using time to moribund condition as the primary endpoint. We will also evaluate RBT/colistin or RIF/colistin combination therapy in mice. Specific Aim 3: Define the breadth of efficacy of RBT Gram-negative and positive bacteria. We will determine the in vitro activity of RBT vs RIF monotherapy against ESBL or carbapenem-resistant A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa, and methicillin-resistant S. aureus by MIC assay (50 isolates each). Lastly, the activity of RBT vs RIF monotherapy against will be evaluated against the previously mentioned bacteria. Impact: Since RFB is already FDA approved, these results will lead to immediate translation to clinical trials to determine if adjunct RFB therapy improves survival of patients infected with A. baumannii. Furthermore, these results may support a fundamental change in screening methodologies to identify novel antibiotics.